Apolipoprotein aiv as an antidiabetic peptide

ABSTRACT

Methods for treating type two diabetes mellitus in a subject in need thereof and pharmaceutical compositions for the treatment of type two diabetes mellitus are disclosed. The methods include administering an effective amount of apolipoprotein A-IV to the subject. The pharmaceutical composition includes apolipoprotein A-IV formulated for administration to a subject for the treatment of type two diabetes mellitus. Also disclosed are methods for substantially restoring glucose tolerance in a subject in need thereof to a normal level and methods for lowering blood glucose levels in a subject in need thereof.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/434,196, filed Jan. 19, 2011, the entire teachings of which areincorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to a method of treating diabetes. Moreparticularly, the present disclosure relates to a method of treatingtype two diabetes mellitus by administering an effective amount ofapolipoprotein A-IV.

BACKGROUND

The occurrence of diabetes is widespread, with approximately 8% of thepopulation in the United States suffering from diabetes. Diabetes is achronic disease characterized by high blood sugar due to the body'sinability to effectively produce and/or use insulin. Diabetes can leadto a variety of physical complications, including but not limited torenal failure, blindness, nerve damage, heart disease, sleep apnea, andceliac disease. For example, in the United States, diabetes is theleading cause of renal failure, blindness, amputation, stroke, and heartattack. Also in the United States, diabetes is the sixth leading causeof death and has been shown to reduce the life expectancy of middle-agedadults by about five to ten years.

The most common form of diabetes is type two diabetes mellitus(hereinafter “T2DM”). T2DM is characterized by hyperglycemia, insulinresistance, β-cell dysfunction, and dysregulated hepaticgluconeogenesis. Persons suffering from T2DM experience a loss ofglucose-stimulated insulin secretion related to the impaired release ofstored insulin granules from β-cells in the first phase of insulinsecretion. In the second phase of insulin secretion, persons sufferingfrom T2DM experience a gradual loss of the ability to activelysynthesize insulin in response to glucose stimuli.

The prevalence of T2DM is increasing and in 2002, T2DM resulted ingreater than $130 billion in health care expenses. As such, newtherapies for effectively treating T2DM are needed.

SUMMARY

The present disclosure is based on the surprising discovery thatapolipoprotein A-IV is an effective anti-diabetic peptide which isintimately involved in the resolution of T2DM. Apolipoprotein A-IV is akey gut hormone which contributes to post-prandial glucose tolerance andacts as a previously unappreciated mediator in the improvement ofglucose tolerance. Accordingly, in one embodiment, methods of treatingT2DM in a subject in need thereof are disclosed. The method comprisesadministering to the subject an effective amount of an apolipoproteinA-IV or a biologically active analogue thereof having at least 90, 95,96, 97, 98 or 99% identity to the apolipoprotein A-IV.

In another embodiment, a pharmaceutical composition comprisingapolipoprotein A-IV is disclosed. The pharmaceutical compositioncomprises an apolipoprotein A-IV or a biologically active analoguethereof having at least 90, 95, 96, 97, 98 or 99% identity to theapolipoprotein A-IV formulated for administration to a subject for thetreatment of T2DM.

In yet another embodiment, a method for substantially restoring glucosetolerance in a subject in need thereof to a normal level is disclosed.The method comprises administering to the subject an effective amount ofan apolipoprotein A-IV or a biologically active analogue thereof havingat least 90, 95, 96, 97, 98 or 99% identity to an apolipoprotein A-IV,for example, by systemic administration of the apolipoprotein A-IV orthe biologically active analogue thereof.

In yet still another embodiment, a method for lowering blood glucoselevel in a subject in need thereof is disclosed. The method comprisesadministering to the subject an effective amount of apolipoprotein A-IVor a biologically active analogue thereof having at least 90, 95, 96,97, 98 or 99% identity to the apolipoprotein A-IV to the subject inneed, for example, by systemic administration. An “effective amount” isas described below and includes about 0.25 to 2 μg/g of the apoA-IV orthe biologically active analogue thereof.

These and other features and advantages of these and other variousembodiments according to the present disclosure will become moreapparent in view of the drawings, detailed description, and claimsprovided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of the embodiments of the presentdisclosure can be better understood when read in conjunction with thefollowing drawings, where like structure is indicated with likereference numerals, and in which:

FIG. 1 is a side perspective view of a device having a reservoir of apharmaceutical composition and a syringe according to an embodiment ofthe present disclosure.

FIG. 2 is a graph of plasma glucose (mg/dL) in male apolipoprotein A-IVknockout and wild-type mice with respect to time (min) for anintraperitoneal glucose tolerance test.

FIG. 3 is a graph of plasma glucose (mg/dL) with respect to time (min)for an intraperitoneal glucose tolerance test in apolipoprotein A-IVwild-type and knockout animals at 16 months of age.

FIG. 4 is a graph of plasma glucose (mg/dL) with respect to time (min)in male apolipoprotein A-IV knockout mice following the intraperitonealadministration of recombinant apolipoprotein A-IV (μg/g) or salinesolution for an intraperitoneal glucose tolerance test.

FIG. 5 is a graph of plasma glucose (mg/dL) with respect to time (min)in apolipoprotein A-IV knockout mice following the intraperitonealadministration of recombinant apolipoprotein A-I or saline solution foran intraperitoneal glucose tolerance test.

FIG. 6 is a graph of insulin secretion (ng/mL) with respect to time(min) in apolipoprotein A-IV knockout mice following the intraperitonealadministration of recombinant apolipoprotein A-I or saline solution.

FIG. 7 is graph of plasma glucose (mg/mL) with respect to time (min) inapolipoprotein A-IV knockout and wild-type mice on a chronicallyhigh-fat diet for an intraperitoneal glucose tolerance test.

FIG. 8 is a graph of plasma glucose (mg/mL) with respect to time (min)in apolipoprotein A-IV knockout mice on a chronically high-fat dietfollowing the intraperitoneal administration of recombinant mouseapolipoprotein A-IV (1 μg/g) or saline solution for an intraperitonealglucose tolerance test.

FIG. 9 is a graph of plasma glucose (mg/dL) with respect to time (h) indiabetic mice following the intraperitoneal administration ofrecombinant mouse apolipoprotein A-IV (1 μg/g) or saline solution for anintraperitoneal glucose tolerance test.

FIG. 10 depicts the results of a Western blot analysis of the level ofserum amyloid A protein component in apolipoprotein A-IV knockout mice,wild-type mice, and apolipoprotein A-I knockout mice.

FIG. 11 is a graph of plasma glucose (mg/dL) in female apolipoproteinA-IV knockout and wild-type mice with respect to time (min) during anintraperitoneal glucose tolerance test (IPGTT).

FIG. 12. is a graph of plasma glucose (mg/dL) with respect to time (min)in wild type mice following the intraperitoneal administration of 1.0μg/g human apolipoprotein A-IV or saline solution during anintraperitoneal glucose tolerance test.

FIG. 13 is a graph of plasma glucose (mg/dL) with respect to time (min)in female wild type mice following the intraperitoneal administration of1.0 μg/g recombinant mouse apolipoprotein A-IV or saline solution duringan intraperitoneal glucose tolerance test.

FIG. 14 is a bar graph showing the effect of 10 mg/g human apoA-IV onhuman islets depolarized by 30 mM KCl and 250 μM diazoxide in thepresence of 3 mM or 20 mM glucose.

FIG. 15 is a protein with the amino acid sequence of full length wildtype human apolipoprotein A-IV (SEQ ID NO. 1).

FIG. 16 is a protein with the amino acid sequence of full length wildtype mouse apolipoprotein A-IV (SEQ ID NO. 2).

FIG. 17 is a protein with the amino acid sequence of full length wildtype human apolipoprotein A-IV with the addition of glycine at theN-terminus (SEQ ID NO. 3).

FIG. 18 is a protein with the amino acid sequence of humanapolipoprotein A-IV showing polymorphic substitutions T347S, Q360H,and/or E165K and the optional addition of glycine, alanine or valine tothe N-terminus (SEQ ID NO. 4).

FIG. 19 is a polynucleotide (SEQ ID NO. 5) encoding full length wildtype human apolipoproteom A-IV.

Skilled artisans appreciate that elements in the figures are illustratedfor simplicity and clarity and are not necessarily drawn to scale. Forexample, the dimensions of some of the elements in the figures may beexaggerated relative to other elements, as well as conventional partsremoved, to help to improve understanding of the various embodiments ofthe present disclosure.

DETAILED DESCRIPTION

The following terms are used in the present application:

As used herein, the term “effective amount” describes the amountnecessary or sufficient to realize a desired biologic effect. Theeffective amount for any particular application may vary depending on avariety of factors, including but not limited to the particularcomposition being administered, the size of the subject, and/or theseverity of the disease and/or condition being treated. In oneembodiment, an “effective amount” is a dose of about 0.25 to 10 μg/g ofan apolipoprotein A-IV or biologically active analogue thereof.Alternatively, an “effective amount of an apoA-IV or a biologicallyactive analogue thereof is about 1 to 10 μg/g, about 0.25 to 2 μg/g, orabout 1 μg/g. An apoA-IV or a biologically active analogue isadministered one time daily. Alternatively, an apoA-IV or a biologicallyactive analogue thereof is administered about 2 times per day. In yetanother alternative, an apoA-IV or a biologically active analoguethereof is administered more than twice a day, for example, three timesper day. In yet another alternative, apoA-IV is administered once everysecond, third, fourth, fifth or sixth day, or once weekly.

As used herein, the term “desired biologic effect” describes reducingthe effects of, counteracting, and/or eliminating a disease orcondition. For example, in the context of T2DM, desired biologic effectsinclude, but are not limited to lowering blood glucose, improvingglucose tolerance, substantially restoring glucose tolerance to a normallevel, improving insulin secretion, and/or substantially restoringinsulin secretion to a normal level.

As used herein, the term “normal level” describes a level that issubstantially the same as the level in a subject who is not in need oftreatment. For example, in the context of treating T2DM, a normal levelof blood glucose is from about 70 mg/dL to about 130 mg/dL before mealsand less than about 180 mg/dL about one to two hours after meals, orfrom about 70 mg/dL to about 100 mg/dL before meals and less than about140 mg/dL about one to two hours after meals. In another example in thecontext of treating T2DM, a normal level of glucose tolerance describesthe ability of the subject to metabolize carbohydrates such that thelevel of blood glucose is from about 70 mg/dL to about 130 mg/dL beforemeals and less than about 180 mg/dL about one to two hours after meals,or from about 70 mg/dL to about 100 mg/dL before meals and less thanabout 140 mg/dL about one to two hours after meals. In still anotherexample in the context of treating T2DM, the normal level of insulinsecretion is the amount required to maintain a normal level of glucosetolerance, wherein the level of insulin secretion is greater than about1 ng/mL about fifteen hours after meals.

In the context of blood glucose level, the term “restore” describeschanging the blood glucose level of a subject to a normal level.Similarly, in the context of glucose tolerance, the term “restore”describes changing the glucose tolerance of a subject to a normal level.Also, in the context of insulin secretion, “restore” describes changingthe insulin secretion of a subject to a normal level.

In the context of apolipoprotein A-IV, the term “biologically activefragment” describes a fragment of apolipoprotein A-IV which is capableof realizing a desired biologic effect in a subject with T2DM. The term“biologically active analogue” describes an analogue of anapolipoprotein A-IV which is capable of realizing a desired biologiceffect in a subject with T2DM. In one example, a desired biologicaleffect is to restore glucose tolerance in apoA-IV knockout mice asdescribed in Example 2. Another example of a desired biological effectis to cause a statistically significant lowering of abnormal glucoselevels in an animal model of T2DM, such as the mouse model described inExample 7.

As used herein, the term “obese” describes a condition in which asubject is well above a normal weight. In one specific example, the termobese describes a condition in which a subject is more than about 20%over their ideal weight and/or has a body mass index of about thirty orgreater than about thirty. In one embodiment, the subject being treatedis obese; in another embodiment, the subject being treated is not obese;and in yet another embodiment, the subject being treated has a normalbody weight.

Embodiments of the present disclosure relate to methods for treatingT2DM in a subject in need thereof and pharmaceutical compositions forthe treatment of T2DM. In one embodiment, a method of treating diabetesis disclosed. In one particular embodiment, a method of treating T2DM ina subject in need thereof is disclosed, wherein the method comprisesadministering an effective amount of an apolipoprotein A-IV (hereinafter“apoA-IV”) or a biologically active analogue thereof to the subject.

In one embodiment, the method of treating T2DM is effective to lowerblood glucose level of a subject. In one particular embodiment, themethod is effective to lower blood glucose level of a subject by about20 to 50%. In a further embodiment, the method is effective to lower theblood glucose level of a subject by about 40%. In still a furtherembodiment, the method is effective to substantially restore bloodglucose level to a normal level.

In another embodiment, the method of treating T2DM is effective tosubstantially restore glucose tolerance of a subject to a normal level.In one particular embodiment, the method is effective to substantiallyrestore glucose tolerance of a subject to a normal level within abouttwo hours after administration of a dose of an apoA-IV or a biologicallyactive analogue thereof. In another embodiment, the glucose tolerance ofa subject is substantially restored to a normal level for about eight totwelve hours.

In yet another embodiment, the treatment is effective to substantiallyrestore insulin secretion to a normal level. In one particularembodiment, the treatment is effective to substantially restore insulinsecretion to a normal level within about two hours after theadministration of a dose of an apoA-IV or a biologically active analoguethereof. In another embodiment, insulin secretion is substantiallyrestored to a normal level for about eight to twelve hours. In stillanother embodiment, the treatment is effective to lower the level ofC-reactive protein.

In one embodiment, an apoA-IV or a biologically active analogue thereofis administered systemically. Systemic administration of the apoA-IV orthe analogue thereof is selected from the group consisting of oral,subcutaneous, intravenous, intramuscular, and intraperitonealadministration.

In another embodiment, a pharmaceutical composition is disclosed. In oneparticular embodiment, the pharmaceutical composition comprises anapoA-IV or a biologically active analogue thereof. In anotherembodiment, the apoA-IV or analogue thereof is formulated foradministration to a subject for the treatment of T2DM. In thisparticular embodiment, a method for treating T2DM in a subject in needthereof is also provided, wherein the method comprises administering aneffective amount of the pharmaceutical composition to the subject.

An “apolipoprotein A-IV” (also referred to herein as “apoA-IV”) refersto mammalian apoA-IV and includes full-length apoA-IV and biologicallyactive fragments of apoA-IV. The full-length human apoA-IV is a 376amino acid protein (SEQ ID NO: 1), the amino acid sequence of which isshown in FIG. 15; the amino acid sequence of full length mouse apoA-IV(SEQ ID NO. 2) is shown in FIG. 16. Also encompassed by the term“apolipoprotein A-IV” is the known analogue in which a glycine is addedto N-terminus of the apolipoprotein A-IV of the full length humansequence (SEQ ID NO. 3, as shown in FIG. 17), and analogues thereofhaving conservative substitutions for the N-terminal glycine (such asalanine and valine). An “apolipoprotein A-IV” also includes polymorphicforms thereof, including the T347S, Q360H, or E165K substitutions to thehuman sequence represented by SEQ ID NO. 1 or the correspondingpositions of SEQ ID NO. 3. As such, “apolipoprotein A-IV” includes theprotein of SEQ ID NO. 4, shown in FIG. 18.

A biologically active analogue of apolipoprotein A-IV has at least 90,95, 96, 97, 98 or 99% identity to an apolipoprotein A-IV. As describedin the previous paragraph, an apolipoprotein A-IV includes full lengthmammalian apolipoprotein A-IV (e.g., human or mammalian), polymorphicforms thereof, the protein of SEQ ID NOS. 3 and 4 and biologicallyactive fragments of any of the foregoing Amino acid variations in thebiologically active analogues preferably have conservative substitutionsrelative to the wild type sequences. A “conservative substitution” isthe replacement of an amino acid with another amino acid that has thesame net electronic charge and approximately the same size and shapeAmino acid residues with aliphatic or substituted aliphatic amino acidside chains have approximately the same size when the total number ofcarbon and heteroatoms in their side chains differs by no more thanabout four. They have approximately the same shape when the number ofbranches in their side chains differs by no more than one Amino acidresidues with phenyl or substituted phenyl groups in their side chainsare considered to have about the same size and shape. Listed below arefive groups of amino acids. Replacing an amino acid residue with anotheramino acid residue from the same group results in a conservativesubstitution:

-   -   Group I: glycine, alanine, valine, leucine, isoleucine, serine,        threonine, cysteine, and non-naturally occurring amino acids        with C1-C4 aliphatic or C1-C4 hydroxyl substituted aliphatic        side chains (straight chained or monobranched).    -   Group II: glutamic acid, aspartic acid and non-naturally        occurring amino acids with carboxylic acid substituted C1-C4        aliphatic side chains (unbranched or one branch point).    -   Group III: lysine, ornithine, arginine and non-naturally        occurring amino acids with amine or guanidino substituted C1-C4        aliphatic side chains (unbranched or one branch point).    -   Group IV: glutamine, asparagine and non-naturally occurring        amino acids with amide substituted C1-C4 aliphatic side chains        (unbranched or one branch point).    -   Group V: phenylalanine, phenylglycine, tyrosine and tryptophan.

An apolipoprotein A-IV or a biologically active analogue thereof can beglycosylated or unglycosylated. The preparation of recombinantunglycosylated human and mouse apolipoprotein A-IV is described inExample 11. The polynucleotide sequence of full length wild type humanapolipoprotein (SEQ ID NO. 1) is shown as SEQ ID NO. 4 in FIG. 18.Apolipoprotein A-IV used in examples 1-10 is unglycosylated. The apoA-IVmay be prepared according to a method known in the molecular biologyfield. For example, apoA-IV may be prepared via traditional molecularcloning techniques.

Apolipoprotein A-IV knockout mice used in the examples were generatedaccording to procedures disclosed in J Lipid Res. 1997 September;38(9):1782-94, the entire teachings of which are incorporated herein byreference.

In one particular embodiment, the pharmaceutical composition may furthercomprise a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers include a wide range of known diluents (i.e.,solvents), fillers, extending agents, binders, suspending agents,disintegrates, surfactants, lubricants, excipients, wetting agents andthe like commonly used in this field. The pharmaceutical composition ispreferably aqueous, i.e., is a liquid formulation, and preferablycomprises pyrogen free water. These carriers may be used singly or incombination according to the form of the pharmaceutical preparation. Theresulting preparation may incorporate, if necessary, one or moresolubilizing agent, buffers, preservatives, colorants, perfumes,flavorings and the like that are widely used in the field ofpharmaceutical preparation.

The apolipoprotein A-IV or biologically active analogue thereof may beformulated into a dosage form selected from the group consisting oftablets, capsules, granules, pills, injections, solutions, emulsions,suspensions, and syrups. The form and administration route for thepharmaceutical composition are not limited and can be suitably selected.For example, tablets, capsules, granules, pills, syrups, solutions,emulsions, and suspensions may be administered orally. Additionally,injections (e.g. subcutaneous, intravenous, intramuscular, andintraperitoneal) may be administered intravenously either singly or incombination with a conventional replenisher containing glucose, aminoacid and/or the like, or may be singly administered intramuscularly,intracutaneously, subcutaneously and/or intraperitoneally.

The pharmaceutical composition of the invention for treating T2DM may beprepared according to a method known in the pharmaceutical field of thiskind using a pharmaceutically acceptable carrier. For example, oralforms such as tablets, capsules, granules, pills and the like areprepared according to known methods using excipients such as saccharose,lactose, glucose, starch, mannitol and the like; binders such as syrup,gum arabic, sorbitol, tragacanth, methylcellulose, polyvinylpyrrolidoneand the like; disintegrates such as starch, carboxymethylcellulose orthe calcium salt thereof, microcrystalline cellulose, polyethyleneglycol and the like; lubricants such as talc, magnesium stearate,calcium stearate, silica and the like; and wetting agents such as sodiumlaurate, glycerol and the like.

Injections, solutions, emulsions, suspensions, syrups and the like maybe prepared according to a known method suitably using solvents fordissolving the active ingredient, such as ethyl alcohol, isopropylalcohol, propylene glycol, 1,3-butylene glycol, polyethylene glycol,sesame oil and the like; surfactants such as sorbitan fatty acid ester,polyoxyethylenesorbitan fatty acid ester, polyoxyethylene fatty acidester, polyoxyethylene of hydrogenated castor oil, lecithin and thelike; suspending agents such as cellulose derivatives includingcarboxymethylcellulose sodium, methylcellulose and the like, naturalgums including tragacanth, gum arabic and the like; and preservativessuch as parahydroxybenzoic acid esters, benzalkonium chloride, sorbicacid salts and the like.

The proportion of the active ingredient to be contained in thepharmaceutical composition of the invention for treating diabetes can besuitably selected from a wide range.

In one particular embodiment, the subject in need of treatment of T2DMis a mammal. The mammal may be selected from the group consisting ofhumans, non-human primates, canines, felines, murines, bovines, equines,porcines, and lagomorphs. In one specific embodiment, the mammal ishuman. In another embodiment, apoA-IV or a biologically active analoguethereof may be administered to a subject for the treatment of T2DMwherein the subject is obese. Alternatively, apoA-IV may be administeredto a subject for the treatment of T2DM wherein the subject is not obese.

Referring to FIG. 1, in yet another embodiment, a device 1 is disclosed.In one embodiment, the device 1 comprises a reservoir 10 of thepharmaceutical composition previously discussed above. In a furtherembodiment, the reservoir 10 comprises a vial 12. The vial 12 may beformed of any material that does not inhibit the function of thepharmaceutical composition. For example, the vial 12 may comprise glassand/or plastic. Additionally, the vial 12 may comprise a pierceableseptum 14 through which the pharmaceutical composition may be removed.In use, the septum 14 of the vial is pierced by the needle 22 of asyringe 20, the pharmaceutical composition is removed by the syringe 20from the vial 12, and the pharmaceutical composition is administered viainjection to a subject in need.

EXAMPLES

The following non-limiting examples illustrate the methods of thepresent disclosure.

Example 1 Glucose Intolerance of ApoA-IV Knockout Mice

Experimental Protocol.

Male apoA-IV knockout (“hereinafter “KO”) mice were obtained. Wild-type(hereinafter “WT”) mice served as controls. ApoA-IV KO and WT mice wereobtained from a colony kept at the University of Cincinnati (Cincinnati,Ohio). ApoA-IV KO and WT mice were fed a chow diet. Prior to performingthe glucose tolerance tests, ApoA-IV KO mice and WT mice were fasted forfive hours. In the glucose tolerance tests, the apoA-IV KO mice and WTmice were injected intraperitoneally with a dose of about 2 mg/g bodyweight of glucose and plasma glucose was measured at about 0, 15, 30,60, and 120 minutes following the injection of glucose. The glucosetolerance tests were performed twice, once at three months of age andagain at sixteen months of age.

Experimental Results.

As shown in FIG. 2, apoA-IV KO mice were glucose intolerant relative tothe WT mice. Specifically, FIG. 2 shows that plasma glucose levels in WTmice were lower than plasma glucose levels in apoA-IV KO mice for twohours following intraperitoneal injection with glucose. While not beingbound by the theory, the implication of these studies was that apoA-IVis necessary for normal glucose homeostasis (at least in males).Moreover, as shown in FIG. 3, apoA-IV KO mice demonstrated an increasedglucose intolerance when tested at sixteen months of age. Specifically,FIG. 3 shows that plasma glucose levels in apoA-IV KO mice tested atsixteen months of age were higher than the plasma glucose levels inapoA-IV KO tested at three months of age. While not being bound by thetheory, the implication of these studies was that glucose tolerance ofapoA-IV KO mice worsens with age.

Experiment with Female Wild Type and ApoA-IV Knockout Mice

Female ApoA-IV wildtype and knockout mice were subjected to the sameintraperitoneal glucose intolerance test as was used for the maleapoA-IV KO and WT mice, as described earlier in this Example 1. Theresults are shown in FIG. 11. Female apoA-IV^(−/−) mice, when challengedintraperitoneally with glucose, have increased plasma glucose levelscompared with female WT animals, but there is no statistical significantdifference. On the other hand, the males have a significant differencebetween WT and KO animals.

Example 2 Restoration of Glucose Tolerance in ApoA-IV Knockout Mice

Experimental Protocol.

Upon demonstrating that apoA-IV KO mice are glucose intolerant, a seriesof extensive studies were performed to determine whether administrationof apoA-IV to apoA-IV KO mice would restore glucose tolerance to anormal level. Specifically, a series of studies were performed todetermine not only the amount of apoA-IV to be administered but also theoptimal time in which to administer apoA-IV prior to conducting glucosetolerance tests.

ApoA-IV male KO mice were injected intraperitoneally with doses of about0.25, 0.5, 1, and 2 μg/g by weight of apoA-IV. ApoA-IV KO mice were alsoinjected intraperitoneally with saline solution to serve as a control.Following injection with mouse apoA-IV or saline solution, glucosetolerance tests were conducted at three months of age as previouslydiscussed. Specifically, glucose tolerance tests were conducted abouttwo hours following injection with apoA-IV or saline solution.Experimental results indicated that the optimal time to restore glucosetolerance in apoA-IV KO mice was to administer apoA-IV about two hoursprior to conducting glucose tolerance tests.

Experimental Results.

As shown in FIG. 4, the administration of apoA-IV to apoA-IV KO micerestored glucose tolerance to a normal level. Specifically, FIG. 4 showsthat plasma glucose levels in apoA-IV KO mice injected with apoA-IV werelower than plasma glucose levels in apoA-IV KO mice injected with salinesolution. Moreover, as shown in FIG. 4, plasma glucose levels in apoA-IVKO mice injected with apoA-IV were the lowest in the apoA-IV KO miceinjected with the highest dosage of apoA-IV; similarly, plasma glucoselevels in apoA-IV KO mice injected with apoA-IV were the highest in theapoA-IV KO mice injected with the lowest dosage of apoA-IV. Accordingly,it was discovered that the degree of improvement of glucose tolerancewas dependent on the dose of apoA-IV administered, with higher dosesresulting in improved glucose tolerance.

Example 3 Specificity of ApoA-IV in Restoring Glucose Tolerance inApoA-IV Knockout Mice

Experimental Protocol.

In order to assess the specificity of apoA-IV, we administeredapolipoprotein AI (hereinafter “apoA-I”) to apoA-IV KO mice. ApoA-I is aprotein made by the small intestinal epithelial cells which also produceapoA-IV. ApoA-I shares many of the functions of apoA-IV. ApoA-IV KO micewere injected intraperitoneally with a dose of 1 μg/g by weight ofapoA-I. ApoA-IV KO mice were also injected intraperitoneally with salinesolution to serve as a control. Following injection with apoA-I orsaline solution, glucose tolerance tests were conducted at three monthsof age as previously discussed. Specifically, glucose tolerance testswere conducted about two hours following injection with apoA-I or salinesolution.

Experimental Results.

As shown in FIG. 5, the administration of apoA-I to apoA-IV KO micefailed to restore or improve glucose tolerance.

Example 4 Mechanism of Restoration of Glucose Tolerance in ApoA-IVKnockout Mice

Experimental Protocol.

In order to assess the mechanism by which ApoA-IV improves glucosetolerance in apoA-IV KO mice, we measured glucose-induced insulinsecretion in apoA-IV KO mice. More specifically, we measuredglucose-induced insulin secretion during glucose tolerance tests atthree months of age as previously discussed. In this study, apoA-IV KOmice were injected intraperitoneally with a dose of about 1 μg/g byweight of mouse apoA-IV two hours prior to conducting the glucosetolerance tests. ApoA-IV KO mice were injected with saline solutionabout two hours prior to conducting glucose tolerance tests to serve asa control.

Experimental Results.

As shown in FIG. 6, phase I insulin secretion was absent in apoA-IV KOmice injected with saline solution. However, as shown in FIG. 6, phase Iinsulin secretion was restored in apoA-IV KO mice when apoA-IV wasinjected intraperitoneally two hours prior to performing the glucosetolerance tests.

Example 5 Efficacy of ApoA-IV in ApoA-IV Knockout and Wild-Type Mice onHigh Fat Diets

Experimental Protocol.

ApoA-IV KO and WT mice were chronically fed a high-fat semi-purified,nutritionally complete experimental diets (AIN-93M) purchased from Dyets(Bethlehem, Pa.) for 10 weeks. The high-fat diets contain about 20 g offat (i.e. about 19 g of butter fat and 1 g of soybean oil to provideessential fatty acids) per 100 g of diet. The apoA-IV KO and WT micewere housed in individual tub cages with corncob bedding in atemperature- (about 22±1° C.) and light- (about 12 h light/12 dark)controlled vivarium. Glucose tolerance tests were performed at threemonths of age as previously discussed. Prior to performing the glucosetolerance tests, apoA-IV KO mice and WT mice were fasted for five hours.In the glucose tolerance tests, the apoA-IV KO mice and WT mice wereinjected intraperitoneally with a dose of about 2 mg/g body weight ofglucose.

Experimental Results.

As shown in FIG. 7, apoA-IV KO mice displayed greater glucoseintolerance relative to the WT mice. Specifically, FIG. 7 shows thatplasma glucose levels in WT mice were lower than plasma glucose levelsin apoA-IV KO mice for two hours following intraperitoneal injectionwith glucose.

Example 6 Restoration of Glucose Tolerance in ApoA-IV Knockout andWild-Type Mice on High Fat Diets

Experimental Protocol.

A series of studies were performed related to the administration ofapoA-IV to apoA-IV KO and WT mice on high-fat diets for 14 weeks atthree months of age (20% by weight of fat, 19% of butter fat and 1% ofsafflower oil). Specifically, apoA-IV KO and WT mice were injectedintraperitoneally with a dose of about 1 μg/g body weight of mouseapoA-IV. ApoA-IV KO and WT mice were also injected intraperitoneallywith saline solution to serve as a control. Following injection withapoA-IV or saline solution, glucose tolerance tests were conducted.Specifically, glucose tolerance tests were conducted two hours followinginjection with apoA-IV or saline solution.

Experimental Results.

As shown in FIG. 8, the administration of apoA-IV in apoA-IV KO micesignificantly improved glucose tolerance. Specifically, FIG. 8 showsthat plasma glucose levels in apoA-IV KO mice injected with apoA-IV werelower than plasma glucose levels in apoA-IV KO mice injected with salinesolution. [the previous sentence is redundant since the next sentencedescribes the same thing. Although the data is not included herein, itwas also discovered that the administration of apoA-IV in WT mice fedchronically a high fat diet also significantly improved glucosetolerance.

Example 7 Restoration of Glucose Tolerance in Mice with T2DM

Experimental Protocol.

In order to confirm that apoA-IV is effective in promoting glucosetolerance in animals with T2DM, heterozygous KK Cg-A/J (hereinafter“Cg-A/J”) mice were obtained from Jackson Laboratories (Bar Harbor,Me.). Cg-A/J mice develop hyperglycemia, hyperinsulinemia, obesity, andglucose intolerance by eight weeks of age. The main cause of diabetes inthese mice is insulin resistance produced by the polygenic interactionswith the A^(y) mutation, which encodes the agouti related protein andantagonist of the melanocortin-IV receptor. The Cg-A/J mice were fedchow diet. Additionally, there was a marked increase in blood glucosefrom ten to fourteen weeks of feeding the chow diet.

At fourteen weeks of age, the Cg-A/J mice were administered either mouseapoA-IV (about 1 μg/g body weight) or saline solution (to serve as acontrol) via intraperitoneal injection. Plasma glucose was thendetermined at about 0, 0.5, 1, 2, 3, 4, 5, 7, 11, and 24 hours.

Experimental Results.

As shown in FIG. 9, apoA-IV has a marked effect in lowering the bloodsugar level of the Cg-A/J mice relative to the saline control. While theCg-A/J mice injected with saline solution maintained a steady plasmaglucose level throughout the 24 hour period of study, the Cg-A/J miceinjected with apoA-IV experienced a decrease in plasma glucose for over10 hours, and, during most of this period, the plasma glucose level wascomparable to the C57BL/6J animals we have been studying. From thisstudy, we conclude that the administration of apoA-IV is effective inlowering the plasma glucose in Cg-A/J mice.

Example 8 Level of Serum Amyloid P Component in ApoA-IV KO, ApoA-I KO,and WT Mice

Experimental Protocol.

A series of studies were performed in related to determining the levelof serum amyloid A protein component (hereinafter “SAP”) in apoA-IV KO,apoA-I KO, and WT mice on atherogenic diets. The apoA-IV KO, apoA-I KO,and WT mice were obtained from the University of Cincinnati. SAP is aserum form of amyloid P component (hereinafter “AP”), a 25 kDapentameric protein first identified as the pentagonal constituent of invivo pathological deposits called amyloid. SAP behaves like C-reactiveprotein in humans. Specifically, the level of plasma SAP in apoA-IV KO,apoA-I KO, and WT mice was determined in apoA-IV KO, apoA-I KO, and WTmice after 12 weeks on an atherogenic diet. The level of plasma SAP wasdetermined via Western blot analysis.

Experimental Results.

As shown in FIG. 10, the level of SAP in apoA-IV KO mice (correspondingto mouse numbers 1, 8, and 10) increased relative to the level of SAP inapoA-I KO mice (corresponding to mouse numbers 28, 29, and 30) and WTmice (corresponding to mouse numbers 19, 20, and 25).

For the purposes of describing and defining the present disclosure it isnoted that the terms “about” and “substantially” are utilized herein torepresent the inherent degree of uncertainty that may be attributed toany quantitative comparison, value, measurement, or otherrepresentation. The terms “about” and “substantially” are also utilizedherein to represent the degree by which a quantitative representationmay vary from a stated reference without resulting in a change in thebasic function of the subject matter at issue.

The above description and drawings are only to be consideredillustrative of exemplary embodiments, which achieve the features andadvantages of the present disclosure. Modification and substitutions thefeatures and steps described can be made without departing from theintent and scope of the present disclosure. Accordingly, the disclosureis not to be considered as being limited by the foregoing descriptionand drawings, but is only limited by the scope of the appended claims.

Example 8 Human ApoA-IV Lowers Blood Glucose Levels in Wild-Type MiceUndergoing Intraperitoneal Glucose Tolerance Testing

Experimental Protocol.

Studies were performed to determine whether administration of humanapoA-IV to wild type mice would affect blood glucose levels in miceundergoing glucose tolerance testing.

Three month old wild type mice were injected intraperitoneally withdoses of about 1 μg/g by weight of human apoA-IV. As a control, anothergroup of wild type mice was injected intraperitoneally with salinesolution. Following injection with human apoA-IV or saline solution,glucose tolerance tests were conducted. Specifically, glucose tolerancetests were conducted about two hours following injection with apoA-IV orsaline solution and after five hours of fasting. Tail blood wascollected and measure by glucometer.

Experimental Results.

As shown in FIG. 12, human apoA-IV was effective in lowering bloodglucose in wild type mice undergoing glucose tolerance testing.

Example 9 Effect of Mouse ApoA-IV in Wild-Type Female Mice UndergoingIntraperitoneal Glucose Tolerance Testing

Experimental Protocol.

Studies were performed to determine whether administration of mouseapoA-IV to female wild type mice would affect blood glucose levels inmice undergoing glucose tolerance testing.

Three month old female wild type mice were injected intraperitoneallywith doses of about 1 μg/g by weight of mouse apoA-IV. As a control,another group of female wild type mice were injected intraperitoneallywith saline solution. Following injection with human apoA-IV or salinesolution, glucose tolerance tests were conducted. Specifically, glucosetolerance tests were conducted about two hours following injection withapoA-IV or saline solution and after five hours of fasting. Tail bloodwas collected and measure by glucometer.

Experimental Results.

As shown in FIG. 13, mouse apoA-IV was effective in lowering bloodglucose in wild type female mice undergoing glucose tolerance testing.

Example 10 Human ApoA-IV Stimulates Insulin Release in Human Islets

High purity human islets were provided by University of Virginia, AxonCells. Islets were cultured in RPMI 1640, containing 10% FBS and 11 mMglucose at 37° C. in a humidified atmosphere of 95% air and 5% CO₂ for48 hours. Four Groups of 50 IEQ islets were then pre-incubated at 37° C.for 1 h in regular KRB (129 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂ 1.2 mMMgSO₄, 1.2 mM KH₂PO₄, 5 mM NaHCO₃, 10 mM HEPES and 0.2% BSA) containing3.0 mM glucose. Islets in the first two groups were then incubated inregular KRB containing 3.0 mM glucose for an hour in the presence orabsence of 10 μg/ml human A-IV and were further incubated with 20 mMglucose for an additional hour in the presence or absence of 10 μg/mlhuman A-IV. Islets in the last two groups were incubated in 30 mM KClKRB (103.8 mM NaCl, 30 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mMKH₂PO₄, 5 mM NaHCO₃, 10 mM HEPES and 0.2% BSA) plus 250 μmol/l diazoxidecontaining 3.0 mM glucose for an hour in the presence or absence of 10μg/ml human A-IV and were further incubated with 20 mM glucose for anadditional hour in the presence or absence of 10 μg/ml human A-IV. Mediawere collected at the end of each one-hour incubation. Insulin levelswere measured by ELISA kit (Millipore).

As can be seen from FIG. 14, when the human islets were maximallydepolarized by 30 mM KCl plus 250 μM diazoxide, 10 μg/ml hA-IV showed asignificant stimulatory effect on insulin secretion.

Example 11 Preparation of Unglycosylated ApoA-IV

Human and mouse apoA-IV cDNA was contained in a pSP65 maintenancevector, and an Afl III restriction site was engineered immediately 5′ ofthe coding sequence for the mature apoA-IV protein. The gene was excisedfrom the maintenance vector and ligated into the pET30 expressionvector. The construct was transfected into E. Coli BL-21 (DE3) cells andgrown in Luria-Bertani cultures supplemented with kanamycin (30 μg/ml)at 37° C. After induction of apoA-IV protein synthesis in the cells, thecells were harvested and sonicated. ApoA-IV protein from the lysate waspurified by His-bind affinity column chromatography and dialysis. Theresultant apoA-IV protein was diluted to a final concentration of 1.0mg/ml in saline.

1-24. (canceled)
 25. A polypeptide, wherein the amino acid sequence ofthe polypeptide is: (SEQ ID NO. 4)    X₁EVSADQVATVMWDYFSQLSNNAKEAVEHLQKSELTQQLNALFQDKLGEVNTYAGDLQKKLVPFATELHERLAKDSEKLKEEIGKELEELRARLLPHANEVSQKIGDNLRELQQRLEPYADQLRTQVNTQAEQLRRQLTPYAQRMERVLRENADSLQASLRPHADX₂LKAKIDQNVEELKGRLTPYADEFKVKIDQTVEELRRSLAPYAQDTQEKLNHQLEGLTFQMKKNAEELKARISASAEELRQRLAPLAEDVRGNLRGNTEGLQKSLAELGGHLDQQVEEFRRRVEPYGENFNKALVQQMEQLRQKLGPHAGDVEGHLSFLEKDLRDKVNSFFSTFKEKESQDKX₃LSLPELEQQQEQX₄QEQQQEQVQMLAPLES

wherein, X₁ is G, A or V; X₂ is E or K; X₃ is T or S; and X₄ is Q or H.26. The polypeptide of claim 25, wherein the amino acid sequence of thepolypeptide is: (SEQ ID NO. 3)    GEVSADQVATVMWDYFSQLSNNAKEAVEHLQKSELTQQLNALFQDKLGEVNTYAGDLQKKLVPFATELHERLAKDSEKLKEEIGKELEELRARLLPHANEVSQKIGDNLRELQQRLEPYADQLRTQVNTQAEQLRRQLTPYAQRMERVLRENADSLQASLRPHADELKAKIDQNVEELKGRLTPYADEFKVKIDQTVEELRRSLAPYAQDTQEKLNHQLEGLTFQMKKNAEELKARISASAEELRQRLAPLAEDVRGNLRGNTEGLQKSLAELGGHLDQQVEEFRRRVEPYGENFNKALVQQMEQLRQKLGPHAGDVEGHLSFLEKDLRDKVNSFFSTFKEKESQDKTLSLPELEQQQEQQQEQQQEQVQMLAPLES.


27. The polypeptide of claim 25, wherein the apolipoprotein A-IV isglycosylated.
 28. The polypeptide of claim 25, wherein theapolipoprotein A-IV is unglycosylated.
 29. The polypeptide of claim 26,wherein the apolipoprotein A-IV is glycosylated.
 30. The polypeptide ofclaim 26, wherein the apolipoprotein A-IV is unglycosylated.
 31. Apharmaceutical composition comprising the polypeptide of claim
 25. 32.The pharmaceutical composition of claim 31, further comprising apharmaceutically acceptable carrier or diluent.
 33. The pharmaceuticalcomposition of claim 32, wherein the pharmaceutical composition is aliquid formulation.
 34. The pharmaceutical composition of claim 33,wherein the pharmaceutical composition is an aqueous formulation. 35.The pharmaceutical composition of claim 34, wherein the aqueousformulation is pyrogen free.
 36. A pharmaceutical composition comprisingthe polypeptide of claim
 26. 37. The pharmaceutical composition of claim36, further comprising a pharmaceutically acceptable carrier or diluent.38. The pharmaceutical composition of claim 37, wherein thepharmaceutical composition is a liquid formulation.
 39. Thepharmaceutical composition of claim 38, wherein the pharmaceuticalcomposition is an aqueous formulation.
 40. The pharmaceuticalcomposition of claim 39, wherein the aqueous formulation is pyrogenfree.